Fig 2: Plasma and PMNs of patients with septic shock at the onset of secondary infection. (A) Plasma ND6 levels of the plasma obtained from patients who had developed secondary infections (second infection (+), n = 3) and secondary infection–negative patients (second infection (−), n = 3). (B) mtDNA levels. (C) Plasma levels of cytokines and chemokines. Each sample was assayed in duplicate. At the onset of secondary infection, plasma ND6 levels in second infection (+) patients (2,521 pg/mL) were still higher than plasma ND6 levels in healthy volunteers (627 pg/mL) and second infection (−) patients (951 pg/mL). Plasma mtDNA levels in second infection (+) patients (2,054 pg/mL) were also higher than in healthy volunteers (14 pg/mL) and second infection (−) patients (143 pg/mL). However, plasma LTB4 levels at the onset of secondary infection were significantly lower in second infection (+) patients (65 pg/mL) than in second infection (−) patients (82 pg/mL), unlike plasma LTB4 levels during the early 72 h period of septic shock. For detailed information, see SI Appendix, Table S8. (D) The chemotaxis of PMNs obtained from healthy volunteers exposed to the plasma of second infection (+) and second infection (−) patients. When PMNs obtained from healthy volunteers were exposed to the plasma of second infection (+) patients, PMN chemotaxis in response to 100 nM fMLF significantly decreased, when compared to PMNs exposed to the plasma of second infection (−) patients (P = 0.001). (E) Representative figures of flow cytometry detecting FPR1 on the PMN membrane and (F) Mean fluorescence intensities. FPR1 expression on the PMN membrane in PMNs exposed to the plasma of second infection (+) patients also decreased compared to PMNs exposed to the plasma of second infection (−) patients (P = 0.030). (G) The chemotaxis of PMNs obtained from second infection (+) and second infection (−) patients in response to 100 nM fMLF. The chemotaxis of PMNs from second infection (+) patients significantly decreased, when compared to PMNs from second infection (−) patients (P = 0.005). (H) Representative figures of flow cytometry detecting FPR1 on the PMN membrane and (I) Mean fluorescence intensities. FPR1 expression on the PMN membrane in PMNs from second infection (+) patients also decreased compared to PMNs obtained from healthy volunteers (P = 0.030), but FPR1 expression of the PMN membrane in PMNs from second infection (−) patients did not decrease significantly. The data are presented as the mean ± SE of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Effects of ND6 pretreatment on bacteria-killing activities of PMNs. (A) Changes in the number of CFUs of S. aureus during the 60 min incubation period with PMNs. Each line represents the mean value of each time point. (B) Calculated S. aureus killing activities of untreated and 100 nM ND6-treated PMNs. Bactericidal activity was calculated by subtracting the number of CFUs of extracellular bacteria and intracellular bacteria in bacteria with PMNs from the number of CFUs of extracellular bacteria in bacteria-only control. (C) Changes in the number of CFUs of E. coli during the 60 min-incubation period with PMNs. (D) Calculated E. coli killing activities of untreated and 100 nM ND6-treated PMNs. ND6 treatment slightly suppressed the bactericidal activity of PMNs both in the PMNs incubated with S. aureus and E. coli, but the difference was not significant. The data are presented as the mean ± SE of three independent experiments.

Fig 4: Microfluidic PMN migration chip assay. (A) Representative figures of microfluidic PMN migration chip experiments. Images were captured at 15 min of chip running from Movie S1 (scale bar, 50 μm length). (B) The number of completely migrated PMNs (chemotactic PMNs) and PMNs entering microchannel entrances in the control medium (CM, DMEM containing 10% FBS) and the sepsis-mimicking medium (SMM). (C) The fraction of chemotactic PMNs among the PMNs entering microchannel entrances. (D) Inhibitory effects of 100 nM ND6 pretreatment on PMN chemotaxis after subtracting chemokinetic PMNs. ND6 pretreatment significantly reduced the fraction of chemotactic PMNs in response to fMLF in both the CM and SMM. Representative figures of chemotactic and chemokinetic PMN movements are shown in SI Appendix, Fig. S13 A and B, respectively. (E) The migrating velocities (μm/second) of PMNs. ND6 pretreatment increased the velocities of PMNs in the CM but not in the SMM. The data are presented as the mean ± SE of three independent experiments. Four different conditions were studied in each experiment, and PMN movements were observed in five fields at random in each chip. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Roles of ND6 in PMN chemotaxis. (A) ER calcium depletion in response to ND6 prestimulation and subsequent fMLF stimulation. Each line represents the mean value of each time point. (B) The AUCs of ER calcium depletion. The white bars represent the mean AUCs for 150 s (AUC150) of ER calcium depletion in response to ND6 prestimulation, and the black bars represent the mean AUC150 in response to fMLF. (C) PMN chemotaxis in response to fMLF after ND6 pretreatment. (D) Gradient PMN chemotaxis in response to ND6 after ND6 pretreatment. (E) Representative figures of flow cytometry detecting FPR1 on the PMN membrane. (F) Mean fluorescence intensities of flow cytometry. The addition of 10 μM CsH completely decreased the number of FPR1 detected on the PMN membrane. Furthermore, 100 nM fMLF significantly decreased FPR1, and ND6 also decreased FPR1 in a dose-dependent manner. (G) Inhibitory effects of ND6 pretreatment on PMN chemotaxis in the sepsis-mimicking medium. In the sepsis-mimicking medium, PMN chemokinesis was significantly increased, and inhibitory effects of ND6 pretreatment on PMN chemotaxis in response to fMLF were weaker than in the control medium: DMEM containing 10% FBS. However, even in the sepsis-mimicking medium, 100 nM ND6 pretreatment significantly suppressed PMN chemotaxis in response to fMLF. (H) FPR1-blockade effects on PMN chemotaxis. In the sepsis-mimicking medium, FPR1-mediated PMN chemotaxis in response to fMLF and ND6 was significantly blocked by pretreatment with an FPR antagonist, CsH (10 μM). In the medium without fMLF or ND6, 0.1% dimethyl sulfoxide (DMSO) was added. The data are presented as the mean ± SE of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.